Human ras oncogene (H-ras, K-ras and N-ras) encoded proteins (p21) have been characterized using monoclonal antibodies against p21 or antioligopeptide antibodies. p21 proteins with 12th amino acid substitutions were grouped into slow moving species as compared to fast moving species of p21 proteins having amino acid substitutions at the 61st position. Analysis of p21 proteins together with diagnostic restriction enzyme analysis of the oncogenes provided a useful method for the detection of site of activation in human ras oncogenes. Although p21 proteins exhibited a heterogenous patterns on SDS-polyacrylamide gels, there appeared to be a conserved mode of post-translational processing. The altered electrophoretic mobilities of p21 proteins as a result of amino acid substitutions probably reflect the confirmational change in the protein which may be responsible for the oncogenic potential of p21 proteins. Transforming efficiency of DNA from a lung carcinoma-derived cell line Hs242 was 10-25-fold lower compared to other tumor cell lines and the Hs242 lines was comprised predominantly of stromal cells of normal appearance. To exclude the possibility that the Hs242 transforming gene arose from minor undetectable fraction of tumor cells, a transforming gene (c-H-ras) was cloned and characterized directly from Hs242 cells. Three positive clones were obtained in LambdaL47 cloning vector and all the clones were positive in NIH/3T3 transfection assay. These results suggest the presence of activated c-H-ras oncogenes in Hs242 cells, although the cells exhibit normal phenotype.